Site-specific fluorescence spectrum detection and characterization of hASIC1a channels upon toxin mambalgin-1 binding in live mammalian cells

Article abstract of DOI:10.1039/c5cc01418b

The synthesis of fluorescent unnatural amino-acid Anap was optimized and the Anap was incorporated into four sites in an acid-pocket or a transmembrane region of human acid-sensing ion channel-1a (hASIC1a). Combinational Anap fluorescence spectra and patc

Full text of DOI:10.1039/c5cc01418b

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Wen, X. Guo,
P. Sun, L. Xiao, J. Li, Y. Xiong, J. Bao, T. Xue, L. Zhang and C. Tian, Chem. Commun., 2015, DOI:
1
0.1039/C5CC01418B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/C5CC01418B
Journal Name
RSCPublishing
COMMUNICATION
Site-Specific Fluorescence Spectrum Detection and
Characterization of hASIC1a Channels upon Toxin
Mambalgin-1 binding in Live Mammalian Cells
Cite this: DOI: 10.1039/x0xx00000x
‡
‡
Received 00th January 2012,
Accepted 00th January 2012
Ming Wen , Xiaoqi Guo , Peibei Sun, Liang Xiao, Juan Li, Ying Xiong, Jin Bao, Tian
* *
Xue, Longhua Zhang , Changlin Tian
DOI: 10.1039/x0xx00000x
www.rsc.org/
The synthesis of fluorescent unnatural amino-acid Anap was mechanical stress. Ion channel regulation has been extensively
optimized and the Anap was incorporated into four sites in investigated using various biophysical methods. Fluorescence
labeling was used to probe conformational changes in the region
acid-pocket or transmembrane region of human acid-sensing
ion channel-1a (hASIC1a). Combinational Anap
surrounding the fluorophore that lead to changes in the
7
microenvironment and hence emission spectra. For in vivo site-
fluorescence spectra and patch-clamp electrophysiology data
illustrated site-specific conformational responses upon toxin
mambalgin-1 binding. This combinational approach can be
used to analyse conformational properties of many different
eukaryotic proteins in their functional states, in a site-
specific manner in live mammalian cells.
specific fluorescence analysis of ion channels, fluorophores can be
coupled to the sulfhydryl group of naturally occurring or introduced
cysteine residues. However, only surface-exposed residues in the
extracellular domains of ion channels can be analyzed using the
8
cysteine-based site-specific fluorophore incorporation method.
Recently, the fluorescent UAA Anap was incorporated into a
+
voltage gated K channel in a site-specific manner to facilitate the
Fluorescent probe labeling of proteins is emerging as a frequently
used method for protein localization, functional characterization,
cell biology and high resolution cellular imaging studies. The
general method involves attaching the target protein to a fluorescent
9
study of the dynamic internal pore opening in oocyte cells .
However, the oocyte system could not provide suitable condition
for structure-function correlation studies of general eukaryotic
proteins (especially soluble proteins or membrane proteins other
than ion channels).
Site-specific incorporation of a small UAA such as Anap can in
principle facilitate the site- and protein-specific characterization of
eukaryotic ion channels in live mammalian cells using combined
fluorescence visualization and confocal microscopy. However,
most of the combinational studies can only provide information of
fluorescence intensity. Despite of high signal intensity (quantum
yield of 0.48 in ethanol), Anap has demonstrated high sensitivity to
1
2
protein or fusion tag specifically coordinating a dye compound.
However, the large size of fluorescent proteins or fusion tags (>20
kDa), and the typical requirements for C- or N-terminal fusion
partners set limits for functional and structural studies of proteins in
3
vitro and in vivo. Recently, an alternative approach was developed
for incorporating a small unnatural amino acid (UAA) (<250 Da)
into the target protein in a site-specific manner, using the
4
orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair. Several
different unnatural amino acids were incorporated into amber
nonsense codon sites, including 7-hydroxycoumarin, dansyl side
chains, and amino-naphthalene derivatives in Escherichia coli,
solvent polarity with a significant shift in emission maximum
max
(λ
em) on transitioning from water (490 nm) to ethyl acetate (420
4b
nm). Therefore, changes in Anap fluorescence spectra, especially
4b, 5
Saccharomyces cerevisiae, and mammalian cells, respectively.
The successful site-specific incorporation of environment-sensitive
-(6-acetylnaphthalen-2-ylamino)-2-amino propanoic acid (Anap)
in target proteins has greatly facilitated functional and structural
max
red- or blue-shifting of λ em, could be used to probe
conformational changes or microenvironment variations in the
vicinity of the incorporated UAA. To this end, Anap flurescence
spectra were collected using an additional prism-based
monochromator prior to emission signal acquisition (Fig. 1A). This
approach might be superior to standard fluorescence intensity
detection, as is often used in voltage clamp fluorometry (VCF)
3
4a
studies of eukaryotic proteins in live mammalian cells , which is
superior to site-specific NMR analysis of proteins’ conformation or
dynamics due to its high sensitivity and easier incorporation in
6
mammalian cell system .
7a
studies on ion channels.
Ion channels are pore-forming membrane proteins that transport
ions across the membrane, and these proteins can be regulated by
Human acid sensing ion channels (hASIC) are proton-gated
stimuli such as voltage, ligands, auxiliary proteins, temperature and cation channels in neuronal system associated with nociception,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 1

ChemComm
Page 2 of 4
View Article Online
COMMUNICATION
C 41
DOI: 10.1039/CJo5Curn01al N8Bame
fear, depression, seizure and degeneration, suggesting roles in pain, Anap incorporation sites were located in the hASIC1a acid-pocket
1
0
12a, 17
neurological and psychiatric disorders . Inhibition of hASIC region (Fig. 2A) and predicted for mambalgin-1 binding.
The
therefore emerges as a new potential therapeutic strategy with huge Anap fluorescence image of CHO cells expressing hASIC1a-
physiological and pharmaceutical significance. Mambalgin-1 is a T236Anap and hASIC1a-D347Anap displayed a blue color in the
cysteine rich, 57 residue polypeptide isolated from the venom of cell envelope under confocal microscopy, which confirmed that the
black mamba snakes, which was demonstrated to abolish pain hASIC1a proteins were correctly trafficked to the cell plasma
11
through inhibition of hASIC in central or peripheral neurons.
membrane (Fig. 2B, 2C). The observed various fluorescence
Recently, the toxin was chemically synthesized and the structure intensities of hASIC1a with Anap incorporation in different cells
12
was determined , however, no detail mechanism studies on were probably due to different transfection efficiency of the
hASIC’s response to the mambalgin-1 binding were reported yet, plasmids. The acquired Anap fluorescence spectra of hASIC1a-
1
2a, 13
except some recent mutagenesis studies
. Here, Anap was site- T236Anap in the CHO membrane region demonstrated a minor
specifically incorporated into hASIC1a expressed in mammalian intensity attenuation upon perfusion of 500 nM mambalgin-1 (Fig.
max
CHO cells to facilitate conformational analysis upon mambalgin-1 2B), but it was difficult to estimate the λ
binding. We started with the Anap synthesis optimization upon relatively large measurement errors. However, λ
shift due to the
em
max
em
red shifting
previous protocols, which was modified to initiate from the readily (from 463 nm to 470 nm) and pronounced intensity attenuations
accessible compound 2-bromonaphthalene (Fig. 1B). Following were observed for hASIC1a-D347Anap upon perfusion of
Friedel-Crafts
bromonaphthalene,
performed that achieved the final product Anap. A total of 8 steps
acetylation
to
generate
2-acetyl-6- increasing mambalgin-1 concentrations (Fig. 2C). Furthermore, a
14
a
Fukuyama-Mitsunobu reaction was sigmoid curve was fitted to the Anap fluorescence intensities at
1
5
max
λ
versus mambalbin-1 concentration (Fig. 2D). The observed
em
and 10 days were required for the entire synthesis, and each step dose-dependent response verified the direct correlation between
was optimized to ensure mild conditions and the highest possible toxin binding and changes in the environment surrounding the
yield. Apart from the relatively low-yielding first step (35%), all D347Anap site.
other steps gave yields between 73–97%, giving a final yield of
With the aid of blue Anap fluorescence images of CHO cells
expressing the labeled channels, Anap fluorescence spectra were
also acquired for the same cells expressing hASIC1a-Y68Anap (Fig.
2
0.7% from 2-bromonaphthalene (for experimental details, see the
Supporting Information).
3
5
A) or hASIC1a-F69Anap (Fig 3C) in the absence or presence of
00 nM mambalgin-1. These Anap incorporation sites are located
1
6-17
in transmembrane helix 1 (TM1; Y68Anap, F69Anap)
(Fig. 3C).
A
pronounced intensity drop was observed in the Anap
fluorescence spectra for hASIC1a-Y68Anap upon toxin perfusion
Fig. 3A), while no obvious Anap fluorescence spectra changes
(
were observed for hASIC1a-F69Anap. The observations indicated
that there might be conformational changes in the Y68Anap site
upon toxin binding, but not in the F69Anap site.
Figure 1. (A) Schematic illustration of the two-photon excitation and spectrum
acquisition using confocal microscopy. A prism-based monochromator was set
up before the photo-multiplier. (B) Synthesis of Anap starting from 2-
bromonaphthalene. Reagents, conditions and yields are shown (a) AcCl, AlCl
PhNO , 100°C, 5 h, 35%; (b) Boc-NH , CuI, DMEDA, K CO , toluene, 110 °C,
0 h, 71%; (c) TFA, DCM, rt, 3 h. 95%; (d) i,o-NsCl, pyridine, DCM, rt,
overnight; ii, N-Trt-Ser-OMe, DIAD, PPh , toluene, rt, overnight, 88% over two
steps; (e) i, TFA, DCM, H O, rt, 4 h; ii, PhSH, K CO , DMF, rt, 3 h, 73% over
two steps; (f) 2 M HCl, 60°C, 8 h, 97%.
3
,
2
2
2
3
3
3
2
2
3
To implement site-specific functional characterization of
hASIC1a in live mammalian cells, fluorescence spectra of Anap-
labeled hASIC1a channels expressed in CHO cells were collected
using two-photon excitation (excitation: 720 nm, two-photon;
emission: 380–550 nm), with Anap incorporations at different sites
of hACIS1a, in the absence or presence of mambalgin-1 (at
different toxin concentrations). Four sites were chosen for the
fluorescent spectra analysis: T236, D347, Y68 and F69. The first Figure 2. Fluorescence spectrum acquisition of Anap incorporation at the acid-
two sites were located in the extracellular domain, close to the acid- pocket region of hASIC1a expressed in CHO cells at pH 7.4. (A) Structural
model of hASIC1a, based on the crystal structure of chicken ASIC1 (PDB:
pocket region which can sense the pH gating or toxin binding,
while the latter two were located in the transmembrane domain,
3
S3W), with the two mutation sites (T236 and D347) and their side-chain
orientations shown. (B) Fluorescence spectra of hASIC1a-T236Anap in the
reflecting the conformational responses of the channel upon ligand absence or presence of 500 nM mambalgin-1, and fluorescence image of the
16
binding . Shown in Figure 2 were the Anap fluorescence spectra recording cell (right, bar scale: 50 ꢀm). (C) Fluorescence spectra of hASIC1a-
D347Anap in the absence or presence of mambalgin-1 at the indicated
for hASIC1a-T236Anap and hASIC1a-D347Anap. Both of the two
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 4
Journal Name
ChemComm
View Article Online
COMMUN01IC41ATION
DOI: 10.1039/C5CC 8B
concentrations. The solid lines on the spectrum and fluorescence image are
fitted to the Weibull equation (right, bar scale: 50 ꢀm). (D) Maximum
fluorescence intensities of hASIC1a-D347Anap with different concentrations of
mambalgin-1, as shown in (C). The solid line was fitted to the Hill equation fit to
estimate the mambalgin-1 dose-dependent response.
dependent IC50 variation of the wild type channel (Fig. 4C)
indicated potential conformational changes at the Y68 site upon the
mambalgin-1 binding. This is consistent with the crystal structure,
which shows that Y68 lines the channel pore, and F69 is a lipid-
facing residue (Fig. 3C).
For the two mutants labeled in the acid-pocket region, the
measured IC50 of hASIC1a-T236Anap (220.65 nM) and D347Anap
In order to correlate the Anap fluorescence spectra of different
UAA incorporation sites, single-cell patch-clamp electrophysiology
studies were conducted. Similarly as wild type hASIC1a, the
normalized current of the four mutants with Anap incorporation
demonstrated pH gating channel conductance, despite exhibiting
different pH sensitivities (Fig. 4A). In hASIC1a with Anap
incorporated at either of the TM1 sites (Y68Anap, F69Anap), there
was a shift in the pH gating mid-point from 6.59 to around 6.15,
whereas the pH gating mid-point of hASIC1a with Anap
incorporated in the acid-pocket region (T236Anap, D347Anap)
were shifted to around 5.60. We were curious as to why similar pH
gating mid-point shifts at the same regions led to different Anap
fluorescence intensity attenuations. To correlate channel functional
and fluorescence-based structural analysis, the channel conductance
at different mambalgin-1 concentrations was measured for the four
Anap-labeled hASIC1a proteins (Fig. 4B). Current attenuations
upon increasing mambalgin-1 were observed for all four mutant
channels (Fig. 4C, 4D).
(
(
128.48 nM) were significantly different from the wild type channel
580.20 nM, pH 5.0). Previous studies indicated that the acid-
pocket region is also the toxin binding site, and mambalgin-1
binding may lead to desensitization, resulting in deceased cation
1
1a
conductance. Therefore, incorporation of Anap in the acid-pocket
region may cause some steric hindrance that disrupts mambalgin-1
binding, leading to different desensitization effects and possibly
even different IC50 values. At the same time, the observed
pronounced Anap fluorescence intensity attenuation of the channel
labeled at the D347Anap site upon increasing mambalgin-1 (Fig.
2
C, 2D) suggests that this site might be in or close to the toxin
binding site. In this case, mambalgin-1 binding could cause a
change in the environment of the D347Anap label, resulting in the
spectral changes (Fig. 2C). Only minor Anap fluorescence intensity
attenuation was observed at the T236Anap site in the absence or
presence of 500 nM toxin (Fig. 2B), indicating that this site is
distant from the toxin binding region.
Figure
4. Electrophysiological characterization of wild type and mutant
Figure 3. Fluorescence spectra acquisition of Anap incorporation at
transmembrane helix 1 of hASIC1a expressed in CHO cells at pH 7.4. (A)
Fluorescence spectra of hASIC1a-Y68Anap in the absence or presence of 500
nM mambalgin-1, and a fluorescence image of the recording cell (right, bar
scale: 50 ꢀm). (B) Fluorescence spectra of hASIC1a-F69Anap in the absence
hASIC1a channels in response to different pH and toxin concentrations. (A)
Channel conductance measurements of wild type or Anap incorporated
hASIC1a channels at different pH. The pH responses were fitted to the Hill
equation. The pH gating mid-points were calculated as 6.59 ± 0.02, 6.13 ± 0.04,
6.20 ± 0.06, 5.60 ± 0.12, 5.66 ± 0.05 for hASIC1a WT (n = 4), F69Anap (n = 5),
Y68Anap (n = 6), D347Anap (n = 4) and T236Anap (n = 4), respectively. (B)
Conductance traces of the hASIC1a-F69Anap, hASIC1a-D347Anap channel
inhibitory effect in the presence of 100 nM and 1000 nM mambalgin-1. To
exemplify the channel attenuation, the channel conductance was measured
under channel open conditions (i.e. hASIC1a-Y68Anap, F69Anap were
measured at pH 6.0, while hASIC1a-T236Anap and D347Anap were measured
at pH 5.0). (C) The dose-response of channel conductance measurements at
or presence of 500 nM mambalgin-1, and
a fluorescence image of the
recording cell (right, bar scale: 50 ꢀm). (C) Structural model of hASIC1a
showing the two mutated sites (Y68 and F69) and their side-chain orientations.
For the two mutants with Anap labeling at TM1, the measured
IC50 of hASIC1a-F69Anap (163.33 nM) was similar as the wild different mambalgin-1 concentrations at pH 6.0. IC50 values were calculated
as 150.87 ± 17.58 nM for hASIC1a-WT (n = 5), 163.33 ± 28.30 nM for
hASIC1a-F69Anap (n = 4), and 90.09 ± 11.02 nM for hASIC1a-Y68Anap (n =
type channel (150.87 nM, pH 6.0), whereas the hASIC1a-Y68Anap
mutant differed markedly (90.09 nM). From previous structural
studies, it is known that the two TM1 sites are further away from
4
). (D) The dose-response of channel conductance measurements at different
mambalgin-1 concentrations at pH 5.0. IC50 values were calculated as 580.20
the toxin binding region, but TM1 is essential for channel ± 190.47 nM for hASIC1a WT (n = 4), 128.48 ± 38.12 nM for hASIC1a-
1
6-17
D347Anap (n = 4) and 220.65 ± 80.70 nM for hASIC1a-T236Anap (n = 5).
opening/closing and for cation conductance.
Therefore, the
correlation between the Anap fluorescence intensity attenuation
upon mambalgin-1 binding (Fig. 3A) and the mambalgin-1 dose
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

ChemComm
Page 4 of 4
View Article Online
COMMUNICATION
C 41
DOI: 10.1039/CJo5Curn01al N8Bame
1
3
M. Salinas, T. Besson, Q. Delettre, S. Diochot, S. Boulakirba, D.
Douguet and E. Lingueglia, The Journal of biological chemistry, 2014,
Conclusions
2
89, 13363.
In a summary, the synthesis of Anap was optimized to make use
of the readily available compound 2-bromonaphthalene, resulting in
an improved yield under mild conditions. Following standard
procedures of UAA incorporation, Anap fluorescence spectra were
1
1
1
4
5
6
R. B. Girdler, P. H. Gore and J. A. Hoskins, J Chem Soc C, 1966, 518.
Z. Xiang and L. Wang, J Org Chem, 2011, 76, 6367.
J. Jasti, H. Furukawa, E. B. Gonzales and E. Gouaux, Nature, 2007,
449, 316.
acquired using an additional prism-based monochromator prior to 17 I. Baconguis and E. Gouaux, Nature, 2012, 489, 400.
emission data collection using two-photon excitation fluorescent
confocal microscopy. With this instrument, changes in protein
conformation or the environment of the labeled sites were probed.
Residues in the acid-pocket and TM-1 regions were investigated in
the absence and presence of mambalgin-1. Anap fluorescence data
were combined with electrophysiological measurements on wild
type and labeled proteins. These results revealed differences in the
conformational changes in the toxin binding and channel pore
regions upon mambalgin-1 binding. Therefore, combinations of the
fluorescence spectra analysis with genetically incorporated small-
size fluorescence UAA and electrophysiology studies can be used
to investigate the molecular mechanisms underlying eukaryotic ion
channel properties in a site-specific manner in live mammalian cells.
Notes and references
Hefei National Laboratory for Physical Sciences at the Microscale and
School of Life Sciences, University of Science and Technology of China,
Hefei 230026
, Anhui, P.R.China. Email: zlhustc@ustc.edu.cn,
cltian@ustc.edu.cn
‡
†
These authors contributed equally to this work
Electronic Supplementary Information (ESI) available: Detailed
experimental methods. See DOI: 10.1039/c000000x/
1
B. N. Giepmans, S. R. Adams, M. H. Ellisman and R. Y. Tsien,
Science, 2006, 312, 217.
2
3
I. R. Correa, Jr., Current opinion in chemical biology, 2014, 20, 36.
(a) C. Stadler, E. Rexhepaj, V. R. Singan, R. F. Murphy, R. Pepperkok,
M. Uhlen, J. C. Simpson and E. Lundberg, Nature methods, 2013, 10,
3
15; (b) Z. Hao, S. Hong, X. Chen and P. R. Chen, Accounts of
chemical research, 2011, 44, 742.
4
5
(a) A. Chatterjee, J. Guo, H. S. Lee and P. G. Schultz, Journal of the
American Chemical Society, 2013, 135, 12540; (b) H. S. Lee, J. Guo,
E. A. Lemke, R. D. Dimla and P. G. Schultz, Journal of the American
Chemical Society, 2009, 131, 12921.
(a) J. Wang, J. Xie and P. G. Schultz, Journal of the American Chemical
Society, 2006, 128, 8738; (b) B. E. Cohen, T. B. McAnaney, E. S. Park,
Y. N. Jan, S. G. Boxer and L. Y. Jan, Science, 2002, 296, 1700.
P. Shi, D. Li, H. Chen, Y. Xiong, Y. Wang and C. Tian, Protein Sci,
6
7
8
2
012, 21, 596.
(a) J. Kusch and G. Zifarelli, Biophysical journal, 2014, 106, 1250; (b)
A. Cha and F. Bezanilla, Neuron, 1997, 19, 1127.
(a) G. Bonifacio, C. I. Lelli and S. Kellenberger, The Journal of general
physiology, 2014, 143, 105; (b) C. J. Passero, S. Okumura and M. D.
Carattino, The Journal of biological chemistry, 2009, 284, 36473.
T. Kalstrup and R. Blunck, Proceedings of the National Academy of
Sciences of the United States of America, 2013, 110, 8272.
9
1
0 (a) K. A. Sluka, O. C. Winter and J. A. Wemmie, Current opinion in
drug discovery & development, 2009, 12, 693; (b) R. Waldmann, G.
Champigny, F. Bassilana, C. Heurteaux and M. Lazdunski, Nature,
1
997, 386, 173.
1
1
1 (a) S. Diochot, A. Baron, M. Salinas, D. Douguet, S. Scarzello, A. S.
Dabert-Gay, D. Debayle, V. Friend, A. Alloui, M. Lazdunski and E.
Lingueglia, Nature, 2012, 490, 552; (b) J. A. Wemmie, R. J. Taugher
and C. J. Kreple, Nature reviews. Neuroscience, 2013, 14, 461.
2
(a) C. I. Schroeder, L. D. Rash, X. Vila-Farres, K. J. Rosengren, M.
Mobli, G. F. King, P. F. Alewood, D. J. Craik and T. Durek,
Angewandte Chemie, 2014, 53, 1017; (b) M. Pan, Y. He, M. Wen, F.
Wu, D. Sun, S. Li, L. Zhang, Y. Li and C. Tian, Chemical
communications, 2014, 50, 5837.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012